Abstract
Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.
Highlights
Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms
When expressed as ectodomain constructs stabilized with SOSIP mutations, trimers based on this clade A sequence can be readily produced at high yields while preserving the native-like, pre-fusion conformation targeted by known broadly neutralizing antibodies[1]
Based on the negative stain EMPEM (nsEMPEM) analysis, we selected polyclonal Fabs isolated from animals Rh.32034 (Grp 1), Rh.33104 (Grp 1) and Rh.33311 (Grp 2) for cryoEM studies
Summary
Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. While BG505 SOSIP trimers have consistently elicited autologous NAb responses in rabbit and macaque animal models[8,10,20,21], that were shown to be protective in macaques[22], they have failed to induce broadly neutralizing responses[23] This is partly due to limited accessibility and immunoquiescence of bnAb epitopes, caused by extensive glycan shielding and sequestration of functionally essential protein domains in the quaternary structure[24]. The antibody response is redirected towards the readily accessible, and, in some cases immunodominant, epitopes that are typically strain-specific Comprehensive mapping of such “off-target” epitopes in BG505 SOSIP and structural characterization of elicited antibodies will provide essential information for engineering the generation of BG505-based immunogens with enhanced on-target reactivity. Twenty-one high-resolution maps of trimer immune complexes with polyclonal Fabs provided detailed insights to the nature of antibody responses at eight unique epitope clusters predominantly targeted in BG505 SOSIP immunogens
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